Method of identifying molecules that regulate FGF activity

ABSTRACT

Disclosed are fibroblast growth factor (FGF) binding and FGF receptor activation, and a method of identifying small molecular weight compounds that interact with FGF to modulate its activity such as, e.g., activators and inhibitors. Illustrative small oligosaccharides, namely di- and tri-saccharides, are shown to be effective modulators of FGF binding and FGF receptor activation.

This invention was made in part with government support under GrantNumber CA 60673 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

The present invention relates to fibroblast growth factor (FGF) bindingand FGF receptor (FGFR) activation. More particularly, the inventionrelates to a method of identifying compounds that interact with FGF andmodulate its activity such as, for example, activators and inhibitors.

(Note: Literature references on the following background information andon conventional test methods and laboratory procedures well known to theordinary person skilled in the art, and such other state-of-the-arttechniques as used herein, are indicated in parentheses and appended atthe end of the specification.)

FGFs regulate a diverse range of physic logic processes such as cellgrowth and differentiation and pathologic processes involvingangiogenesis, wound healing and cancer (1).

FGFs utilize a dual receptor system to activate signal transductionpathways (2-5). The primary component of this system is a family ofsignal-transducing FGF receptors (FGFRs) that contain an extracellularligand-binding domain and an intracellular tyrosine kinase domain (1).

The second component of this receptor system consists of heparan sulfate(HS) proteoglycans or related heparin-like molecules which are requiredin order for FGF to bind to and activate the FGFR (3,4).

Although the mechanism by which heparin/HS activates FGF is unknown,heparin, FGF and the FGFR can form a trimolecular complex (3).

Heparin/HS may interact directly with the FGFR linking it to FGF (6).Furthermore, heparin/HS can facilitate the oligomerization of two ormore FGF molecules, which may be important for receptor dimerization andactivation (3). There are no pharmacologic agents that were previouslyknown to modulate the activity of FGFs.

Heparin/HS is a heterogeneously sulfated glycosaminoglycan that consistsof a repeating disaccharide unit of hexuronic acid and D-glucosamine. Ithas been previously reported that, at a minimum, highly sulfated octa-(3) or decasaccharide (7) fragments derived from heparin are requiredfor FGF to bind to the FGFR. However, preparation of these heparinfragments produces mixtures of isomers and chemically modifies theoligosaccharide ends (8). Furthermore, size-fractionated heparin maycontain individual molecules with distinct biological properties.

Accordingly, it would be desirable to determine the mechanism by whichheparin/HS activates FGF and, further, to identify compounds thatinteract with FGF and modulate its activity.

In U.S. Pat. No. 5,270,197, various systems are described for assayingthe ability of a substance to bind to a high-affinity heparin-bindinggrowth factor (HBGF) receptor, e.g. an FGF receptor. The disclosure ofsaid patent is incorporated herein by reference.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, a method is provided formodulating the activity of heparin/HS-dependent mitogenesis whichcomprises subjecting a biological fluid which contains FGF to amitogenesis-modulating amount of a small oligosaccharide having from 2to about 6 saccharide units.

The biological fluid can be any fluid which contains or is compatiblewith the FGF, e.g., an aqueous solution of FGF, conditioned cell culturemedium, serum or plasma, or a physiologically acceptable solution of theFGF. Agents that modulate the activity of FGF are useful in woundhealing, angiogenesis and cancer treatment.

According to another embodiment of the invention, a method is providedfor identifying a compound that interacts with FGF to thereby modulateits activity comprising subjecting said compound to one or more of thefollowing assay procedures:

(a) testing the ability of the compound as a modulator of FGF-dependentmitogenesis on FGF receptor-expressing cells and comparing against themitogenic activity of heparin or sucrose octasulfate;

(b) testing the ability of the compound to compete with the binding of¹²⁵ I-heparin to FGF;

(c) testing the ability of the compound to enhance or inhibit thebinding of FGF to a soluble or cell surface FGF receptor;

(d) testing the ability of the compound to enhance or inhibit FGFdimerization;

(e) testing the ability of the compound to enhance or inhibit FGFreceptor dimerization; and

(f) testing the ability of the compound to enhance or inhibit FGFbinding to heparin or heparin immobilized on a gel matrix.

The gel matrix for the heparin immobilization can be, e.g., an agarosesuch as SEPHAROSE.®

Compounds that can thus be identified as modulators of FGF activity are,e.g., organic compounds, compounds that contain carbohydrate moietiessuch as oligosaccharides and polysaccharides and compounds containingheparin-like structures. These compounds preferably are small molecularweight compounds of up to about 1000 Daltons.

Initially, to determine the molecular mechanism by which heparin/heparansulfate (HS) activates FGF, small non-sulfated oligosaccharides foundwithin heparin/HS were assayed for activity. These oligosaccharides aredi- and tri-saccharides which are synthetic and isomerically purecompounds and do not contain any modified sugar residues (9). Thechemical structures of illustrative small oligosaccharides are set forthin Table 1, below.

                                      TABLE 1                                     __________________________________________________________________________    Heparin/HS oligosaccharides.                                                  Name Mw  Structure.sup.#                                                      __________________________________________________________________________    Di-1 433.3                                                                             α-L-IdoA-(1→4)-α-D-GlcNAc-1→OMe            Di-2 493.2                                                                             α-L-IdoA-(1→4)-α-D-GlcNSO.sub.3 -1→OMe              1                                                                    Di-3 433.3                                                                             β-D-GlcA-(1→4)-α-D-GlcNAc-1→OMe             Di-4 493.2                                                                             β-D-GlcA-(1→4)-α-D-GlcNSO.sub.3 -1→OMe      Di-5 433.3                                                                    D-GlcNAc-(1→4)-β-D-GlcA-1→OMe                              Tri-1                                                                              631.4                                                                             β-D-GlcA-(1→4)-α-D-GlcNAc-(1→4)-β-D             -GlcA-1→OMe                                                   Tri-2                                                                              691.2                                                                             α-L-IdoA-(1→4)-α-D-GlcNSO.sub.3 -(1→4)-             β-D-GlcA-1→OMe                                           Tri-3                                                                              631.4                                                                             α-L-IdoA-(1→4)-α-D-GlcNAc-(1→4)-β-             D-GlcA-1→OMe                                                  __________________________________________________________________________     .sup.# IdoA, iduronic acid; GlcA, glucuronic acid; GlcN, glucosamine; Gal     galactose; aManOH, 25-anhydro-D-mannitol; Ac, acetate; Me, methyl.       

The most preferred of these small oligosaccharides are Tri-1 and Tri-3.They are active at concentrations comparable to heparin and are1000-fold more active than sucrose octasulfate (SOS) in an assay whichmeasures the proliferation of FGFR-expressing BaF3 cells (3) in thepresence of basic FGF (bFGF). Four disaccharides, Di-2, 3, 4 and 5, showintermediate mitogenic activity with bFGF, with D-3 and D-4 being themost active of the disaccharides.

The most preferred small oligosaccharides, Tri-1 and Tri-3, alsostimulate the proliferation of FGFR-expressing BaF3 cells in thepresence of acidic FGF (aFGF).

These results with small oligosaccharides having from 2 to about 6saccharide units were unexpected since they are considerably smallerthan the smallest heparin/HS oligosaccharides previously shown toactivate FGF, namely octa- (3) and decasaccharides (7).

DETAILED DESCRIPTION OF THE INVENTION

While the specification concludes with claims particularly pointing outand specifically claiming the subject matter regarded as forming thepresent invention, it is believed that the invention will be betterunderstood from the following detailed description of preferredembodiments of the invention taken in conjunction with the appendeddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graphical representation which shows the mitogenic activityof synthetic oligosaccharides. Activation of bFGF mitogenic activity asmeasured by ³ H-thymidine incorporation into FGFR1-expressing BaF3 cellstreated with 150 pM bFGF and increasing concentrations of the indicatedoligosaccharides is shown. FIG. 1B. Activation of aFGF mitogenicactivity determined as above in the presence of 250 pM aFGF; these dataare representative of at least two independent experiments.

FIG. 2, in four parts, FIGS. 2A, 2B, 2C AND 2D, is a graphicalrepresentation which shows the biochemical properties of synthetic.oligosaccharides.

FIG. 2A: Binding of oligosaccharides to bFGF as determined bycompetition with ¹²⁵ I-heparin (30).

The IC₅₀ s (50% competition for ¹²⁵ I-heparin binding to bFGF are: 69ng/ml for heparin, 11.7 μg/ml for Tri-1, 5.8 μg/ml Tri-3 and 93 μg/mlfor SOS (sucrose octasulfate).

FIG. 2B: Binding of ¹²⁵ I-bFGF to FRAP (3), a soluble receptor, in thepresence of the indicated oligosaccharide: *, no heparin added; heparin,50 ng/ml; the remaining oligosaccharides (Di-1 to SOS) were added at aconcentration of 2 μg/ml. FGFs were labeled by the Chloramine T methodas previously described (31).

FIG. 2C: Acidic FGF binding to FRAP; FIG. 2D:, basic FGF binding toFRAP. ¹²⁵ I-FGFs were incubated with FRAP in the presence of increasingconcentrations of heparin or Tri-3; Binding assays were performed aspreviously described (3).

FIG. 3, in four parts, FIGS. 3A, 3B, 3C and 3D, shows the crystalstructure of bFGF complexed with synthetic oligosaccharides.

FIG. 3A: Electron density for Tri-3 ligand in sites 2/2' at 2.2 Åresolution.

Thick lines indicate the ligand atoms. Thin lines indicate contour linesof electron density at 1.8σ above the mean density, in a map calculatedusing the coefficients (2|Fo|-|Fc|)exp(-iα_(c)) where |Fo| is theobserved structure factor amplitude, and |Fc| and α_(c) are theamplitudes and phases calculated from the model and adjusted by addingthe structure factor of the solvent (14).

FIG. 3B: Ribbon diagram (32) of bFGF bound to Di-3 molecules, shown withsolid black bonds and labeled according to the sites to which they bind.

The label prime refers to symmetry-related molecules. β strands areshown as arrows. The notation used is according to (17) with strandslabeled from β1 to β12 and loops labeled with the numbers of thesecondary structures they join.

Only secondary structures participating in the Di-3/bFGF interaction areindicated. However, the location and features of the Tri-3 binding sitesare essentially the same.

FIG. 3C: Stereodiagram of sites 1 and 1' with the Tri-3 ligand at adimer interface.

Tri-3 is shown with thick solid black bonds.

Sugar rings are labeled A, B, C, with A indicating iduronic acid, B,N-acetyl glucosamine, and C, O-methyl glucuronic acid.

Medium-thick and thin lines indicate amino acid atoms involved in site 1and 1', respectively.

Dotted lines indicate hydrogen bonds.

Atoms and amino acids involved in hydrogen bonds are indicated by aprime label when the bond involves atoms of site 1'.

The oligosaccharide in site 1 is within a pocket of high positivepotential that includes primarily amino acids of the 10-11 and 11-12loops (see FIG. 3B for notation). Site 1' makes contacts with regions ofthe structure that include the 5-6 loop and the β4 strand.

FIG. 3D: Stereodiagram of sites 2 and 2' with the Tri-3 ligand at adimer interface.

Notation is as in FIG. 3C. Site 2 consists of regions of the structurethat include the β8 strand and the 4-5 loop.

Site 2' consists of a largely hydrophobic platform (strands β6 and β7)flanked by positively charged residues Arg 72 and Lys 86 on one side andresidues Arg 81 and Lys 77 on the other side.

FIG. 4 shows the bFGF dimerization in the presence of heparinhexadecasaccharide (HS-16) or synthetic oligosaccharide Tri-3. 670 nMbFGF and 3×10⁵ cpm ¹²⁵ I-bFGF were incubated with the indicatedconcentration of (μg/ml) Tri-3 or HS-16. Crosslinking andelectrophoresis were performed as previously described (3). Dimer (45kD) band intensities were quantified by scanning densitometry andplotted above each lane. Molecular weight markers (kD) are shown at theright.

FIG. 5 is a schematic diagram of the restriction enzyme map of theplasmid MIRB-FR1, which is an 9.59 Kb expression vector for FGFreceptor-1, a cell surface receptor. In this vector:

MoLTR is the Molony murine leukemia virus long terminal repeat;

mFGFR1 is the FGF receptor-1 open reading frame;

emc IRES is the internal ribosome entry sequence;

NeoR is the neomycin resistance gene;

SV40polyA is the late polyA addition site of the SV40 virus; and

BSSK(+) is the bluescript (pBS) SK (+) vector (Stratagene Inc.)

Restriction enzyme sites are shown on the periphery by theirconventional abbreviations.

FIG. 6 shows the dimerization of FGF receptors induced by FGF-2 andheparin-trisaccharide (Tri-3). ¹²⁵ I-bFGF(2×10⁶ cpm) was incubated with4×10⁶ BaF3-FGFR1 cells. Binding media (DMEM/0.1% BSA) was supplementedwith the indicated concentration of heparin or Tri-3.(*) 200 ng/mlunlabeled FGF-2 added to binding media; Cells were washed once with thesame media used for binding, and once with PBS. Crosslinking was asdescribed previously (27).

Crosslinked receptors were electrophoresed on a 5% SDS polyacrylamidegel under reducing condition, and visualized by autoradiography. Thelower band corresponds to receptor monomers crosslinked to FGF-2. Theupper band corresponds to receptor dimers crosslinked to each other andto FGF-2. Molecular weights (kD) are shown at the right.

In order to illustrate the invention in further detail, the followingspecific laboratory examples were carried out with the resultsindicated. Although specific examples are thus illustrated, it will beunderstood that the invention is not limited to these specific examplesor the details therein.

EXAMPLES

Materials and Methods

Basic fibroblast growth factor (bFGF) was obtained from Scios Nova.Acidic fibroblast growth factor (aFGF) was obtained from Merck & Co.,Inc.

Heparin/HS-derived di- and tri-saccharides were obtained from PharmaciaAB.

FGFR3-expressing BaF3 cells have been described previously (27).

FGFR1-expressing BaF3 cells can be made by transfecting the expressionplasmid MIRB-FR1 (FIG. 5) into BaF3 cells and selecting with theantibiotic G418 (geneticin) at about 600 μg/ml for 10 days and pickingcolonies.

Individual colonies are FGF- and heparin-dependent. The plasmid MIRB-FR1(FIG. 5) is constructed analogously to previously published procedures(27) for preparing FGFR3 expression vectors (see FIG. 5A in Ref. 27),except that the FGFR1 gene is used instead of the FGFR3 gene.

The relationship of the FGFR1 gene and the Molony murine leukemia viruslong terminal repeat (LTR) is identical to that previously published(3). See also U.S. Pat. No. 5,270,197, FIG. 11A, for the construction ofanother suitable plasmid, namely mFR1, which contains the Molony virusLTR, and can be transfected into Ba3F cells and selected with G418.

The FGFR1-expressing BaF3 cell line was used to assay syntheticheparin/HS molecules as potential activators or inhibitors ofheparin/HS-dependent mitogenesis by an assay procedure publishedpreviously (3). Such cell lines, which express FGRF1, require heparin/HSand FGF for growth (3). The ability of heparin/HS-derived di- andtri-saccharides to enhance FGF-FRAP binding in vitro was measured bythis assay.

The structures of di- and tri-saccharides which were thus assayed areshown in Table 1, above. Their synthesis was published previously (9).

The ability of these small oligosaccharides to compete with the bindingon ¹²⁵ I-heparin to FGF was also assayed. Heparin iodination was carriedout by previously published procedure (30). Heparin binding to FGF wasdetermined by incubating 4 nM FGF with ¹²⁵ I-heparin and competitorpolysaccharide. Complexes were immunoprecipitated with 1:250 dilutionsof monoclonal antibody DG2 (DuPont/Merck) (for bFGF) or a polyclonalantibody (for aFGF), and protein A SEPHAROSE (Sigma Chemical Co.).Washing and quantitation was carried out by procedures previouslypublished for soluble receptor-binding assays (3).

The crystal structure of complexes between bFGF and biologically activedi- and trisaccharides was determined. Four. binding sites wereobserved, sites 1, 1', 2 and 2'. Sites 2 and 2' have not been observedpreviously. The ability of these di- and trisaccharides to bind the2/2'sites on bFGF may contribute to their biological activity.

Small crystals of complexes of bFGF (25 mg/ml) and Di-3 or Tri-3 (1 to1.3 ratios) were obtained at 20° C. (33) in 15% or 17.5% (w/v),respectively, polyethylene glycol (average Mr=3350), 0.1M HEPES (pH6.8), and subsequently macroseeded repeatedly in fresh drops of the samesolution. Both complexes form triclinic crystals with one molecule inthe asymmetric unit (a=30.9, b=33.3, c=34.7, α=87.6, β=85.4, γ=76.4).Structure determination was carried out as in (34,35) with the modelgenerated by (18).

The oriented model was then refined by simulated annealing andleast-squares optimization, as in (37). Data from 40 Å to 2.2 Å wereused.

Examination of a map generated using the coefficients(|Fo|-|Fc|)exp(-iα_(c)) showed density that could accommodate saccharidemolecules in both sites 1 and 2.

Subsequent incorporation of the structure factor of the solvent resultedin improved density. Model building and correction was carried out withthe programs O (38,39) and turbo (40).

Least-squares refinement of the saccharide-FGF structure was carried outwith X-PLOR (36), with stereochemical parameters for Di-3 and Tri-3taken from the topology and parameter files provided for pyranosidesugars by the X-PLOR package

The final R-factor is 22.0% and 22.8% for the Tri-3/bFGF and Di-3/bFGFstructures, respectively, with good stereochemistry. Analysis of thetemperature factors indicated that the occupancy of the saccharides islikely to be partial. Occupancy of the di- and trisaccharide was set to0.5, which results in temperature factors that are comparable to thosefor side-chains observed to coordinate the ligands.

Results

The FGFR1-expressing BaF3 cell line, which expresses FGFR 1, requiresheparin/HS and FGF for growth (3). This cell line was used to assaysynthetic heparin/HS molecules as potential activators or inhibitors ofheparin/HS-dependent mitogenesis (3).

Two trisaccharides, Tri-1 and Tri-3, are active at concentrationscomparable to that of heparin (FIG. 1, Table 2). The thirdtrisaccharide, Tri-2 (containing a glucosamine N-sulfate), and fourdisaccharides, Di-2, 3, 4, and 5, show intermediate mitogenic activitywith basic FGF (bFGF) whereas the disaccharide Di-1 and the threetetrasaccharides examined demonstrate no activity in this assay.

Sucrose octasulfate (SOS), a highly charged molecule thought tostabilize and activate bFGF (10), was also examined. Heparin, Tri-1, andTri-3 are greater than 1000-fold more active than SOS forFGFR1-expressing BaF3 cell growth. Di-3 and 4 are 55-fold more active inthis assay.

Unlike previously examined fragments of heparin, several of thesesynthetic heparin/HS molecules are non-sulfated, yet they still havebiological activity. Furthermore, they are considerably smaller than thesmallest heparin/HS oligosaccharide previously shown to activate FGF(3).

Because several of these oligosaccharides are non-sulfated, interactionswith the carbohydrate backbone of heparin/HS appear to be sufficient forbiological activity. Furthermore, the large differences in activityobserved between closely related oligosaccharides suggest that theseinteractions are highly specific.

Two of the three trisaccharides (Tri-1 and Tri-3) stimulateproliferation of FGFR1-expressing BaF3 cells in the presence of aFGF.However, their potency relative to that of heparin is less than thatobserved with bFGF (FIG. 1B). These data demonstrate that thestructure-function relationship between heparin/HS and aFGF is similarto that of bFGF and suggests that recognition of the structural featuresof heparin/HS is a conserved property of FGFs and not specific to asingle ligand.

FGFs have a high affinity for heparin (K_(d) 10⁻⁹ M) (11). The abilityof the synthetic oligosaccharides to compete with the binding of ¹²⁵I-heparin to FGF was assayed (12). Heparin, oligosaccharides Di-3, Di-4,Tri-1, and Tri-3, and the related molecule, SOS, all bind aFGF and bFGF(FIG. 2A, Table 2) (9).

Heparin, Tri-1 and Tri-3 bind FGF with an affinity higher than that ofSOS. Di-3, Di-4 and SOS bind FGF with similar affinity, whereas Di-2 andTri-2 bind bFGF with an affinity less than that of SOS. The number ofhydrogen bonds in the crystal structures of Di-3 and Tri-3 complexedwith FGF (see below) correlate well with these data. The relativebinding affinities also correlate well with the mitogenic activity ofthese molecules. However, on the basis of the relative affinities forbFGF (Table 2), Di-2, Tri-1, and Tri-3 show higher than expectedmitogenic activity. Thus, factors other than direct binding to bFGF maycontribute to the biological activity of these molecules.

Binding of bFGF to a soluble FGFR-alkaline phosphatase fusion protein(FRAP) or to a cell-surface FGFR is enhanced by heparin/HS or byheparin-derived oligosaccharides (8 to 12 sugar residues) (3,4). Theability of heparin/HS-derived di- and tri-saccharides to enhanceFGF-FRAP binding in vitro was measured (3). These studies demonstratethat Di-3, Di-4, and Tri-3 enhance bFGF-FRAP binding (FIG. 2B).

Furthermore, the binding of either aFGF (FIG. 2C) or bFGF (FIG. 2D) toFRAP increases in a dose-dependent manner. At high concentrations ofTri-3, the amount of ¹²⁵ I-bFGF bound to FRAP reproducibly exceeds thatwith heparin (FIG. 2D).

The observation that small di- and trisaccharides can enhance FGF-FRAPbinding suggests that multiple binding sites along a single heparin/HSchain (beads on a string model) are not essential for biologicalactivity, and that the occupancy of a relatively small heparin/HSbinding site(s) on FGF may be sufficient to activate FGF.

The inability of Tri-1 to enhance FGF-FRAP binding suggests that severalmechanisms may mediate FGF receptor activation. Clearly the substitutionof iduronic acid for a glucuronic acid at the non-reducing end of thesaccharide is sufficient to discriminate between different modes ofaction.

Heparin/HS may stabilize a ternary complex by binding directly to theFGFR (6) in addition to FGF. To investigate this model, FRAP wasincubated with ¹²⁵ I-heparin (12). No significant binding (>2 fold overbackground) between ¹²⁵ I-heparin and FRAP could be detected. However,when bFGF (up to 4 nM) was added to this binding reaction, ¹²⁵ I-heparinbinding was increased up to 25.5-fold over background (13). These dataand the observation that molecules as small as disaccharides werebiologically active suggest that the mechanism by which heparin/HSactivates the FGFR results from a primary interaction between the FGFRand a complex of heparin/HS and FGF.

To further evaluate the mechanism by which heparin/HS activates FGF andto establish a framework for the rational design of drugs that modulatethe activity of FGF, the crystal structure of complexes between bFGF andbiologically active di- and trisaccharides (14) was determined (FIG.3A). A view of a molecule of bFGF with bound Di-3 reveals four bindingsites (FIG. 3B). Similar observations were made with the Tri-3/bFGFcomplex (FIGS. 3, C and D). Bound- and apo-FGF structures superimposewith a root-mean-square deviation in C.sup.α positions of 0.26 Å. It istherefore unlikely that a conformational change in FGF is involved inits mechanism of activation by heparin.

Two ligand molecules were observed in the crystal structure. Each ligandcontacted two symmetry-related FGF molecules thus defining two pairs ofbinding sites: 1, 1' and 2, 2' (FIG. 3B). Site 1 is similar to thatobserved for SOS bound to aFGF (15) and is also the site where sulfateions are located in the bFGF apo-structure (16-18). Twelve hydrogenbonds, as defined in (19), form between FGF and Tri-3 at site 1. Incomparison, only three hydrogen bonds form at site 1' (FIG. 3C). Thesedata suggest that sites 1 and 1' are not equivalent in terms of bindingaffinity and therefore are unlikely to be involved in FGFoligomerization. Site 1' most likely results from crystal packingforces.

Sites 2 and 2' (FIG. 3, B and D) have not been observed previously. Thispair of sites is symmetry-related and consequently located very close toa crystal packing interface. In contrast to sites 1 and 1', bothsymmetry-related FGF molecules make extensive contact with Tri-3 atsites 2 (11 hydrogen bonds) and 2' (11 hydrogen bonds). Therefore, eachof these sites is likely to bind ligand with high affinity and each canbe considered an independent binding site. However, the average hydrogenbond length between Tri-3 and site 2' is 0.35 Å shorter than betweenTri-3 and site 2.

In addition, when a more stringent definition of the hydrogen bond wasused (20), eight hydrogen bonds were observed between Tri-3 and 2'compared to only four between Tri-3 and site 2. This indicates that site2' may have greater affinity than site 2, for Tri-3. In the crystalstructure, site 2 of one FGF molecule and site 2' of a symmetry-relatedFGF molecule are brought together by a single oligosaccharide molecule.Such contacts may be responsible for the oligomerization of FGF (seecrosslinking experiments below).

Several other putative heparin/HS binding sites on bFGF have beensuggested (21, 22); however, no density is observed at these sites foreither Di-3 or Tri-3. In contrast to SOS, which has. minimal biologicalactivity and occupies a single site on the aFGF molecule, multiplebinding sites for Di-3 and Tri-3 are observed. The capability of di- andtrisaccharides to bind several sites on FGF may be a requirement foractivity. Additionally, these sites may represent a path followed byheparin/HS polysaccharides between two FGF molecules complexed in afunctional dimer.

The biological relevance of the potential dimer interface at site 2/2'is of significant interest since crosslinking studies demonstrate thatdi- and trisaccharides can induce FGF oligomerization. It is believedthat inhibition of the FGF receptor dimerization (FIG. 6) cansignificantly influence the activity of FGF which, in turn, can beuseful in cancer therapy.

Di-3 and Tri-3, like the highly sulfated heparin hexadecasaccharide(HS-16), can induce FGF dimers as well as higher order oligomers (FIG.4) (3). However, there are notable differences between the syntheticoligosaccharides and HS-16. The optimal concentration for dimerizationactivity induced by Tri-3 (35 μg/ml) is approximately 10-fold greaterthan that of HS-16 (3.9 μg/ml).

At concentrations 10- to 20-fold greater than these levels, the amountof dimerization seen with the hexadecasaccharide approaches basallevels. However, the amount of dimerization seen with the di- (13) ortrisaccharide (FIG. 4) remains elevated. High ratios of HS-16 to FGF mayfavor a stoichiometry of several heparin oligosaccharides per FGF. Underthese conditions, FGF dimerization may be inhibited sterically by therelatively large heparin molecule (HS-16). High ratios of di- ortri-saccharide to FGF would not be expected to sterically inhibit FGFdimerization.

The dimerization of FGF receptors induced by FGF-2 andheparin-trisaccharide (Tri-3) is shown in FIG. 6. ¹²⁵ I-bFGF(2×10⁶ cpm)was incubated with 4×10⁶ BaF3-FGFR1 cells. Binding media (DMEM/0.1% BSA)was supplemented with the indicated concentration of heparin orTri-3.(*) 200 ng/ml unlabeled FGF-2 added to binding media; Cells werewashed once with the same media used for binding, and once with PBS.Crosslinking was as described previously

Crosslinked receptors were electrophoresed on a 5% SDS polyacrylamidegel under reducing condition, and visualized by autoradiography. Thelower band corresponds to receptor monomers crosslinked to FGF-2. Theupper band corresponds to receptor dimers crosslinked to each other andto FGF-2.

The data presented here demonstrate that both non-sulfated di- andtrisaccharides are biologically active in several FGF-dependent assaysand suggest that FGF can specifically recognize structural features ofthe non-sulfated carbohydrate backbone of heparin/HS, independent ofionic interactions with highly charged sulfate groups. However, becauseheparin is more active than low-sulfated heparin (4), it is likely thationic interactions could further stabilize this interaction. The lack ofactivity of Tri-2, a compound that only differs from Tri-3 (the mostactive compound tested) in having a N-sulfate group on the glucosamineresidue, suggests that N-sulfated regions of heparin/HS may not beinvolved in FGFR activation.

Substitution of an N-sulfate in the Tri-3/bFGF crystal structuredemonstrates repulsive interactions between the sulfate group andglutamic acid 96 in site 2.

Several studies demonstrate that 2-O-linked sulfate groups on thehexuronic acid residues of heparin/HS may be important for optimalactivity (23-26).

Sulfation of our synthetic oligosaccharides at the 2-O position mayfurther increase their affinity for bFGF and their biological activity.

The small size of the synthetic heparin/HS molecules suggests thatlinkage of multiple FGFs by heparin/HS in a "beads on a string model" isnot an essential component of the mechanism of FGFR activation. Amechanism is suggested herein in which heparin/HS induces FGF dimerswhich in turn form stable complexes with FGF receptor moleculesfacilitating receptor dimerization. Recent binding studies, with the useof distinct members of the FGF family, suggest that an FGFR may containmultiple, partially overlapping binding sites that involve bothimmunoglobulin-like domains II and III (27). These data are consistentwith FGFR molecules interacting with homo- or heterodimers of FGF.

                  TABLE 2                                                         ______________________________________                                        Glycosaminoglycan cofactor activity.                                                   Mitogenic activity‡                                                           bFGF binding*                                             ______________________________________                                        heparin    1.0 ± 0.5 1.0                                                   Di-2       69.5 ± 9.6                                                                              >1340.6                                               Di-3       26.0         1224.5                                                Di-4       24.7         1224.5                                                Di-5       463.6        >1340.6                                               Tri-1      1.2 ± 0.2 169.3                                                 Tri-2      470.6 ± 25.6                                                                            >1340.6                                               Tri-3       3.2         84.1                                                  SOS        1380.8 ± 30.6                                                                           1340.6                                                ______________________________________                                         ‡Relative concentration (wt/vol.) required to incorporate 2        × 10.sup.4 cpm .sup.3 Hthymidine into F32 cell DNA (3), ±            standard deviation.                                                           *Relative affinity (based on IC50 values (wt/vol.)) for heparin binding t     bFGF. Calculations based on molecular weights give similar ratios,            assuming that the average molecular weight for heparin is 16,000 and that     there are 15 FGF binding sites per heparin/HS molecule (29).             

Various other examples will be apparent to the person skilled in the artafter reading the present disclosure without departing from the spiritand scope of the invention. It is intended that all such other examplesbe included within the scope of the appended claims.

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12. Heparin iodination was as in (30). Heparin binding to FGF wasdetermined by incubating 4 nM FGF with ¹²⁵ I-heparin and competitorpolysaccharide. Complexes were immunopredpitated with 1:250 dilutions ofmonoclonal antibody DG2 (for bFGF) or a polydonal antibody (for aFGF),and protein A sepharose (Sigma). Washing and quantitation was aspreviously described for soluble receptor binding assays (3).

13. D. M. Ornitz, unpublished data.

14. Small crystals of complexes of bFGF (25 mg/ml) and Di-3 or Tri-3 (1to 1.3 ratios) were obtained at 20° C (33) in 15% or 17.5% (w/v),respectively, polyethylene glycol (average Mr=3350), 0.1M Hepes (pH6.8), and subsequently macroseeded repeatedly in fresh drops of the samesolution. Both complexes form triclinic crystals with one molecule inthe asymmetric unit (a=30.9, b=33.3, c=34.7, 0c=87.6, β=85.4, γ=76.4).Structure determination was carried out as in (34, 35) with the modelgenerated by (18). The oriented model was then refined by simulatedannealing and least-squares optimization, as in (37). Data from 40 Å to2.2 Å were used. Examination of a map generated using the coefficients(|Fo|-|Fc|)exp(-iα_(c)) showed density that could accommodate saccharidemolecules in both sites 1 and 2. Subsequent incorporation of thestructure factor of the solvent resulted in improved density. Modelbuilding and correction was carried out with the programs O (38, 39) andturbo (40). Least-squares refinement of the saccharide-FGF structure wascarried out with X-PLOR (36), with stereochemical parameters for Di-3and Tri-3 taken from the topology and parameter files provided forpyranoside sugars by the X-PLOR package (36). The final R-factor is22.0% and 22.8% for the Tri3/bFGF and Di-3/bFGF structures,respectively, with good stereochemistry. Analysis of the temperaturefactors indicated that the occupancy of the saccharides is likely to bepartial. Occupancy of the di- and trisaccharide was set to 0.5, whichresults in temperature factors that are comparable to those forside-chains observed to coordinate the ligands.

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28. C. Svahn, A. Ansari, T. Wehler, unpublished data.

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41. We thank D. McEwen and S. Mathews for discussion, A. Chellaiah, T.Opera, J. Xu and M. Weurffel for help. bFGF and antibody DG2 were giftsfrom J. Abraham (Scious Nova) and W. Herblin (DuPont/Merk),respectively. aFGF and anti-aFGF antisera was a gift from K. Thomas(Merck). Fluoresceinated heparin was a gift from C. Parish (AustralianNational University, Canberra, Australia). SOS was from Bukh Meditec.Heparin was from Hepar Inc. This work was supported in part by grantsfrom the NIH (CA60673), Monsanto-Searle, and Washington UniversityMedical School.

What is claimed is:
 1. A method of modulating the activity ofheparin/HS-dependent mitogenesis comprising subjecting a biologicalfluid which contains FGF to a mitogenesis-modulating amount of a smalloligosaccharide having from 2 to about 6 saccharide units.
 2. The methodof claim 1 in which the oligosaccharide is selected from the groupconsisting of Tri-1 which isβ-D-GlcA-(1→4)-α-D-GlcNAc-(1→4)-β-D-GlcA-1.fwdarw.OMe,Tri-3 which isα-L-IdoA-(1→4)-α-D-GlcNAc-(1→4)-β-D-GlcA-1.fwdarw.OMe, Di-2 which isα-L-IdoA-(1→4)-α-D-GlcNSO₃ -1→OMe, Di-3 which isβ-D-GlcA-(1→4)-α-D-GlcNAc-1→OMe, and Di-4 which isβ-D-GlcA-(1→4)-α-D-GlcNSO₃ -1→OMe.
 3. The method of claim 1 in which theFGF is bFGF.
 4. The method of claim 1 in which the FGF is bFGF and theoligosaccharide isTri-1 which isβ-D-GlcA-(1→4)-α-D-GlcNAc-(1→4)-β-D-GlcA-1.fwdarw.OMe or Tri-3 which isα-L-IdoA-(1→4)-α-D-GlcNAc-(1→4)-β-D-GlcA-1.fwdarw.OMe.
 5. The method ofclaim 1 in which the FGF is aFGF and the oligosaccharide isTri-1 whichis β-D-GlcA-(1→4)-α-D-GlcNAc-(1→4)-β-D-GlcA-1.fwdarw.OMe or Tri-3 whichis α-L-IdoA-(1→4)-α-D-GlcNAc-(1→4)-1→3-.beta.-GlcA-1→OMe.
 6. The methodof claim 1 in which the mitogenesis is enhanced.
 7. The method of claim1 in which the mitogenesis is inhibited.